1. Field of the Invention
This invention relates to new continuous cell lines from embryonic tissues of ticks (Acari Ixodidae); the use of such cells lines for replicating selected microorganisms; and the use of the replicated microorganisms for diagnosis, prophylaxis and control of diseases in vertebrate animals caused by infections of such microorganisms.
2. Description of the Prior Art
Tissue culture and cloning of pure cell strains is an established art, dating back to the in vitro growth and differentiation or nerve cells from an embryo by Harrison in 1909. Many cells lines of arthropods of the class Insecta have successfully been established, but relatively few of the class Arachnida. In particular, few cell lines of the order Acari (Mites and Ticks) and very few of the family Ixodidae (Ticks) are known. For example, Hink in "Invertebrate Tissue Culture, Research Applications", Acad. Press, N.Y., 1976, Pp. 319-369, stated that only 5 cell lines from 3 species of Acarina were known, as contrased with 24 cell lines from 15 species of Lepidoptera and 63 cell lines from 21 species of Diptera. There are various reasons for this, including the extremely delicate microbiology of cells, which is evidenced by extreme sensitivity to culture culture medium and conditions. As a result of this sensitivity, culture media and conditions suitable for cells of a closely related family such as Parasitidae (Dung Beetle Mites) may not also be suitable for Ixodidae (Ticks).
Ticks, because of their complex life cycles in which one or more blood meals are required for each feeding stage, are the most important vectors of infectious disease to vertebrate animals and are second only to mosquitoes as vectors of disease to man. Among the diseases transmitted by ticks are hemorrhagic fevers and other viroses, rickettsioses, anaplasmosis, piroplasmosis, spirochaetosis, tularemia and filariasis. In vitro cultures of tick tissues and cells have obvious benefits for the study of the causal agents of tickborne disease. The few continuous lines of tick cells that have been established are difficult to manage because of slow growth and the fastidious nature of the cells. Such cell lines were disclosed by Varma, Pudney, and Leake, "The Establishment of Three Cell Lines from the Tick Rhipicephalus appendiculatus (Acari: Ixodidae) and Their Infection with some Arboviruses", J. Med. Entomol., 11:698-706, (1975); and by Bhat and Yunker, "Establishment and Characterization of a Diploid Cell Line from the Tick Dermacentor parumapertus Neumann (Acarina: Ixodidae)", J. Parasitol., 63:1092-1098 (1977). The cell line of Bhat and Yunker, 1977, above, has accidentally been extinguished by toxic culture medium and bacterial contamination of frozen stocks.
The general technique for culturing embryonic tick cells used for the cell lines of this invention was disclosed by Yunker and Meibos in "Culture of Embryonic Tick Cells (Acari: Ixodidae)", Tissue Culture Association (TCA) Manual, Vol. 5, No. 1, 1979. Similar disclosure will be found in Bhat and Yunker, 1977, above, and in Pudney, Varma, and Leake, "Establishment of Cell Lines from Ixodid Ticks", TCA Manual, Vol. 5, No. 1, 1979.
The general use of tissue cell lines for the culture or replication of pathogenic microorganisms is well established, although the use of arthropod vector cell lines for culturing zoonotic disease agents is relatively rare. Due to the biochemical nature of microorganisms, especially viruses and rickettsias, specific microorganisms can not be cultured in all cell lines, even in all cell lines from the same order.
Buckley, in "Invertebrate Tissue Culture, Research Applications", Acad. Press. New York and London, (1976), Pp. 201-32 (Chapter 12--Arboviruses and Toxoplasma gondii in Diptera Cell lines), discloses replications of various arthropod vector viruses in mosquito cell lines. She explicitely recognizes marked innate differences between individual cell lines (p. 202) and notes, for example, that several tick viral isolates have failed to propagate in Aedes (mosquito) cell lines (p. 205) while Ganjam virus, isolated from both mosquitoes and ticks, multiplied in the Aedes albopictus cell line but not in the Aedes aegypti cell line (p. 204).
Also similarly, Vaughn in "Invertebrate Tissue Culture, Research Applications", Acad. Press, New York and London (1976) at Pp. 295-303 (Chapter 15--The Production of Viruses for Insect Control in Large Scale Cultures of Insect Cells) discusses the development of insect cell lines and notes that, for example, cell lines from Heliothis zea are not capable of complete replication of the nuclear polyhedrosis virus obtained from the Heliothis zea itself.
From the above, it should be apparent that the ability of a given cell line to replicate successfully a given microorganism, especially a virus or rickettsia, cannot be predicted from results in cell lines of different species. In fact, because of cellular adaptation, even cell lines from the same species may not be equally useful for microorganism replication.
Viruses are "submicroscopic entities capable of being introduced into specific living cells and of reproducing in living cells only" (Luria, General Virology, 1st ed., Wiley, New York, 1953), which have more recently been further defined as "entities whose genomes are elements of nucleic acid that replicate inside living cells using the cellular synthetic machinery and causing the synthesis of specialized elements that can transfer the viral genome to other cells" (Luria, et al., General Virology, 3rd ed., Wiley, New York, 1978).
An important group of vector-borne disease agents are the arthropod-borne viruses (arboviruses), most of which are togaviridae or bunyaviridae. In most cases, the arboviruses are maintained in nature by a vector-host cycle that does not include humans. Five families of blood-sucking animals (Culicidae or mosquitoes, Ceratopogonidae or midges, Psychodidae or sand flies, Ixodidae or hard-shell ticks, and Argasidae or soft-shell ticks) are common vectors for the viruses, and various vertebrates, generally wild mammals and birds, serve as host and reservoirs. The virus is maintained only if balanced populations of hosts and vectors exist, and if they are in contact with each other. The persistence of a virus in nature is governed by the arthropod vector rather than the host, since, typically the arthropod is not damaged by the virus.
It has been noted as one of the most interesting properties of the arboviruses, that they multiply both in a vertebrate host at temperatures up to 39.degree. C. or in insects and insect cell cultures at lower temperatures (Hurlbut and Thomas, "The Experimental Host Range of the Arthropod-borne Animal Viruses in Arthropods", Virology, Vol. 12, Pp. 391-407, 1960; and Pfefferkorn and Shapiro, "Reproduction of Togaviruses in Comprehensive Virology", Fraenkel-Conrab and Wagner, ed. Plenum Press, New York, 1974, Pp. 17-230).
Rickettsiae are intracellular parasites that are small gram-negative bacteria which multiply by growth and fission, possess a bacterial-type cell wall which includes a rigid mucopeptide layer, and have an autonomous energy producing system. They are constrained by synthetic and enzymatic deficiencies to intracellular parasitism. The pathogenic rickettsia are primarily parasites of arthropods, and are occasionally also pathogenic for vertebrates. Rickettsial diseases of man vectored by lice, ticks, mites, etc., include epidemic typhus, Rocky Mountain spotted fever, South American spotted fever, fievre boutonneuse, Q fever, tsutsugamushi disease (scrub typhus), trench fever, and rickettsialpox, and before transmission to man, may be reservoired by rats, wild rodents, field and house mice, cattle, sheep, goats, or man itself, depending upon the particular causal organism.
Patents which appear to relate to this invention are as follows.
U.S. Pat. No. 3,709,782 discloses a heteroploid feline embryonic cell line, the growth of viruses thereon, and vaccines containing such viruses. The disclosed medium is Eagle's Basal Medium with other ingredients, but without Liebovitz's L-15. Moreover, there is no disclosure of non-vertebrate cell lines.
U.S. Pat. No. 4,021,302 is very similar to U.S. Pat. No. 3,709,782, except that the cell line is substantially diploid.
U.S. Pat. No. 4,040,905 discloses a cell line of diploid cells from vertebrates, suitable for virus vaccine production.
U.S. Pat. No. 2,658,021 discloses a production method for vertebrate animal tissue cell lines.
U.S. Pat. No. 3,647,633 discloses an apparatus for the continuous production of microorganisms and plant and animal cell lines.
U.S. Pat. No. 4,017,631 discloses a process for producing continuous tumoral cell lines in vitro.